光诱导对人视网膜色素上皮细胞自噬和凋亡的影响

doi:10.3760/cma.j.cn115909-20210406-00137

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摘要

研究人视网膜色素上皮（RPE）细胞光损伤中自噬与凋亡之间的关系，并探讨自噬抑制剂3甲
基腺嘌呤（3MA）对光诱导下RPE细胞自噬和凋亡的影响。方法：实验研究。将体外培养的人RPE细

胞（ARPE-19）随机分为12 h对照组、12 h模型组、12 h 3MA组以及24 h对照组、24 h模型组、24 h

3MA组。对照组采用锡纸包裹避光培养；模型组接受光照刺激；3MA组中加入3 mmol/ml 3MA后接

受光照刺激，以自制三基色LED（发光二极管）冷光灯作为光源，在（16 500±500）lx光照强度下照

射细胞12 h和24 h。透射电子显微镜观察每组细胞超微结构，流式细胞仪测定细胞存活率，激光共

聚焦结合倒置荧光显微镜定性、定量分析自噬-溶酶体形态及荧光强度，Western blot法检测Beclin 1、

LC3和P62自噬相关蛋白表达量。数据采用单因素方差分析。结果：在光照12 h后，ARPE-19细胞自

噬水平可以抵抗光损伤，降低细胞凋亡率（F=931.53，P<0.001）；光照24 h后细胞自噬水平超过其正

常调节范围，细胞凋亡率升高，差异有统计学意义（F=1156.67，P<0.001）；使用3MA可抑制光诱导

的ARPE-19细胞过度自噬，降低细胞凋亡率（F=2.856，P=0.027），进而保护细胞以应对光损伤。结论：

ARPE-19自噬水平随光照时间而变化；随着光照时间延长，细胞凋亡率升高。抑制细胞的过度自噬

可以保护ARPE-19细胞应对光损伤。

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关键词 ：年龄相关性黄斑变性, 视网膜色素上皮细胞, 光损伤, 3甲基腺嘌呤, 自噬, 凋亡

Abstract：

Objective: To study the effect of light damage on the relationship between autophagy and apoptosis in

human retinal pigment epithelial (RPE) cells and to explore the effect of 3-methyladenine (3MA), an

autophagy inhibitor, on light-induced autophagy and apoptosis in RPE cells. Methods: This experiment

studied human RPE cells (ARPE-19) that were cultured in vitro and randomly divided into the following

groups: 12 h control group, 12 h model group, 12 h 3MA group; 24 h control group, 24 h model group,

and 24 h 3MA group. Tinfoil wrap was used in the control group to avoid light. The model group was

stimulated by light, and the 3MA group received light stimulation after adding 3 mmol/mL 3MA. A custom

made three-primary color LED (light-emitting diode) cold light lamp was used as the light source, and the

cells were irradiated for 12 h and 24 h under 16 500±500 lux of light intensity. A transmission electron

microscope was used to observe cell ultrastructure, flow cytometry was used to measure cell viability,

a confocal laser combined with an inverted fluorescence microscope was used for the qualitative and

quantitative analysis of autophagy-lysosome morphology and fluorescence intensity, and the Western blot

method was used to detect the expression of Beclin 1, LC3 and P62 autophagy-related proteins. The data

were analyzed by one way analysis of variance. Results: The autophagy level of ARPE-19 cells can resist

light damage and reduce the apoptosis rate under 12 hours of light (F=931.53, P<0.001); after 24 hours of

light, the autophagy level of cells exceeded its normal regulation range, and the apoptosis rate increased

(F=1156.67, P<0.001). The difference was statistically significant. The use of the autophagy inhibitor 3MA

inhibited the light-induced excessive autophagy of ARPE-19 cells, reduced the rate of apoptosis (F=2.856,

P=0.027), and protected cells against light damage. Conclusions: The level of ARPE-19 autophagy

changes with the amount of time to light exposure. The cell apoptosis rate increases with prolonged light

exposure. Inhibiting cell autophagy can protect ARPE-19 cells from light damage.

Key words： age-related macular degeneration retinal pigment epithelium cell light damage 3-methyladenine autophagy apoptosis

收稿日期: 2021-04-06

PACS:

基金资助:国家自然科学基金（82060885）；宁夏自然科学基金（2020AAC03172）；宁夏高等学校科学研究项目（NGY2018-99）

通讯作者: 俞洋（ORCID：0000-0001-8024-4969），Email：20070030@nxmu.edu.cn

引用本文:

高园园1，2 李娟1 雒玉3 李武军2 俞洋1. 光诱导对人视网膜色素上皮细胞自噬和凋亡的影响[J]. 中华眼视光学与视觉科学杂志, 2021, 23(10): 728-735. Yuanyuan Gao1, 2, Juan Li1,Yu Luo3,Wujun Li2,Yang Yu1. Effects of Light Induction on Autophagy and Apoptosis of Human Retinal Pigment Epithelial Cells. Chinese Journal of Optometry Ophthalmology and Visual science, 2021, 23(10): 728-735. DOI: 10.3760/cma.j.cn115909-20210406-00137

链接本文:

https://www.cjoovs.com/CN/ 10.3760/cma.j.cn115909-20210406-00137 或 https://www.cjoovs.com/CN/Y2021/V23/I10/728

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